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Peroxidase isozymes in two developmental stages in nine strains of drosophila melanogaster

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Title:
Peroxidase isozymes in two developmental stages in nine strains of drosophila melanogaster
Creator:
Blomer, Allison
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English
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vi, 37 leaves : illustrations ; 29 cm

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Subjects / Keywords:
Isoenzymes ( lcsh )
Microbiology ( lcsh )
Isoenzymes ( fast )
Microbiology ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 31-32).
General Note:
Submitted in partial fulfillment of the requirements for the degree of Master of Arts, Department of Integrative Biology, 1986.
Statement of Responsibility:
by Allison Blomer.

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University of Colorado Denver
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Auraria Library
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17800826 ( OCLC )
ocm17800826

Full Text
PEROXIDASE ISOZYMES IN TWO DEVELOPMENTAL
STAGES IN NINE STRAINS OF DROSOPHILA MELANOGASTER
by
Allison Blomer
B.A., University of Minnesota, 1980
A thesis submitted to the Faculty of the Graduate
School of the University of Colorado in partial
fulfillment of the requirements for the degree of
Master of Arts
Department of Biology
1986
1
$ v


This thesis for the Master of Arts degree by
Allison Blomer
has been approved for the
Department of
Biology
by
Gerald Audesirk
Date


Blomer, Allison (M.A., Biology)
Peroxidase Isozymes in Two Developmental Stages in Nine Strains
of Drosophila melanogaster
Thesis directed by Professor Linda K. Dixon
Peroxidase isozymes were demonstrated in nine distinct
strains of Drosophila melanogaster by zymograms produced by
agarose gel electrophoresis. Developmental changes were studied
by using homogenates of third instar larvae, early pupae, and
middle pupae.
Of the four isozymes detectable by this method, there
is a fairly consistent developmental change in the patterns
observed. The transition from third instar larva to early pupa
was associated with a shift in the isozyme banding patterns.
This shift was from the presence of isozymes #3 and #4 in larvae
to isozymes #1 and #2 in early pupae (isozyme #1 being least
anodic). Isozyme activity decreased in the middle pupae.
However, there was variability in the isozyme patterns
within strains and between strains. The differences between
strains were not as obvious as the developmental differences.
The most striking finding was that of the isozyme patterns of
the completely homozygous strain, CL55. The majority of the
zymograms, (86%), of third instar larvae of strain CL55 showed
only isozymes #3 and #4. This is in contrast to the eight other
strains which showed predominantly isozyme bands #1 and #3.


IV
CONTENTS
CHAPTER
I. INTRODUCTION ...................................... 1
II. MATERIALS AND METHODS............................... 9
Drosophila Stocks and Homogenates................ 9
Procedure........................................ 10
III. RESULTS......................................... . 16
IV. DISCUSSION......................................... 21
BIBLIOGRAPHY............................................ 31
APPENDIX
A. RAW DATA FOR TABLE 3.............................. 33
B. RAW DATA FOR TABLE 4
37


V
TABLES
Table
1. Description and Source of Drosophila melanogaster
Stocks.............................................. 10
2. Summary of the Reagents used in this Procedure. . 11
3. Relative Percentage of each Isozyme within a Pattern
Determined by Densitometric Scans: Third Instar
Larvae.............................................. 18
4. Relative Percentage of each Isozyme within a Pattern
Determined by Densitometric Scans: Early Pupae. . 20


VI
FIGURES
Fi gure
1. Transition of Isozymes seen in Third Instar Larvae
and Early Pupae Illustrated by Densitometric Scans. 17
2. Zymograms Example Isozyme Patterns of Third
Instar Larvae and Early Pupae ........................ 23


CHAPTER I
INTRODUCTION
Peroxidase is a class of enzymes which catalyze the
oxidation of many organic compounds:
AH2 + H202 2H20 + A,
where AH2 is a hydrogen donor (the reducing agent) and A is
its oxidized form. The peroxidases are ferri-hematin compounds
and are a special category of oxidoreductases. All peroxidases
have the common ability of reducing hydrogen peroxide to water.
Peroxidases are further identified according to the substrate
upon which the enzyme acts or according to its site of action.
Thus, there are many types of peroxidase.
Peroxidases occur widely in plant and animal tissues.
Some examples of peroxidases are: NAD-peroxidase, NADP-peroxidase,
cytochrome-c peroxidase, glutathione peroxidase (protection of
erythrocytes), lactoperoxidase (secretory protein in saliva and
milk), myeloperoxidase (found in neutrophilic leukocytes),
eosinophil peroxidase (found in eosinophils), epidermal peroxi-
dase (cuticle formation in insects), thyroid peroxidase (syn-
thesis of thyroid hormones), and prostaglandin hydroperoxidase
(synthesis of prostaglandins). Horseradish peroxidase is the
most studied plant peroxidase. The physical characteristics


2
and reactivities of peroxidases of different origin vary to a
considerable degree.
The various peroxidases are located throughout the
particular organism. Glutathione peroxidase is found in the
cytosol and mitochondrial matrix space (Sunde and Hoekstra,
1980). Myeloperoxidase is contained in cytoplasmic granules and
released into the extracellular space (Matheson et a!., 1981).
Thyroid peroxidase has been localized on the endoplasmic reti-
culum of the rat thyroid gland cells (Edwards and Morrison,
1976). The peroxidase associated with hard cuticle formation
in some insects has been found in cisternae of the rough endo-
plasmic reticulum, in vesicles of Golgi complexes and in
secretory vesicles (Locke, 1969). In Drosophila melanogaster,
peroxidase is found in certain tissues of the digestive tract,
Malphigian tubules, reproductive tract, fat body, brain and
flight muscle of both males and females (LiBassi, 1981).
The molecular properties of peroxidase vary as much as
their functions and locations. Plant peroxidases have molecular
weights about one half that of animal peroxidases. For example,
horseradish peroxidase has a molecular weight of 40,000, lacto-
peroxidase has a molecular weight of 93,000, and myeloperoxidase
has a molecular weight of 118,000. Plant peroxidases generally
show a monomeric character (Espino and Vasquez, 1982). Myelo-
peroxidase appears to consist of two identical subunits and thus
is a dimer (Matheson et al., 1981). Glutathione peroxidase is
known to be a tetramer with four subunits (Board, 1983). These


3
varying molecular properties play a major role in characterizing
the enzyme in the organism.
There are many known and proposed functions of peroxidase.
Two major functions known in plants are the oxidation of the
plant hormone auxin (indole-3-acetic acid) (Espino and Vasquez,
1982) and the catalysis of crosslinks between aromatic alcohols ;
to form a cell wall component, lignin (Stafford and Bravinder-
Bree, 1972). A similar function is seen during the formation
of insect exoskeleton. Peroxidase catalyzes the formation of
crosslinks between tyrosine residues in resilin (Locke, 1969).
The antimicrobial action of peroxidases is another major
functional category. Three known peroxidases in this category
are myeloperoxidase, eosinophil peroxidase and lactoperoxidase.
The microbicidal activity of these peroxidases is carried out
both in the extra-cellular space and within phagocytic vacuoles
(Matheson et al., 1981). The leukocytes undergo a brief meta-
bolic burst during and after phagocytosis, which generates
hydrogen peroxide, upon which bactericidal activity depends.
Peroxidases also serve as essential enzymes in the
synthesis of certain hormones. Thyroid peroxidase catalyzes
the iodination of tyrosines in the synthesis of thyroid hor-
mones (Lehninger, 1975). Prostaglandin hydroperoxidase reduces
peroxide groups to yield prostaglandins (Cavallo, 1976).
Reducing hydrogen peroxide to water is a common function
of all peroxidases and is an important line of defense against
free radical formation in the cell. The free radical theory of


4
aging purports that free radicals (highly reactive chemical
species with unpaired electrons), particularly the superoxide
radical, exist as a normal consequence of cellular metabolism
and also contribute significantly to abnormal reactions and
aging (Harman, 1956).
Membranous components of cells, particularly the un-
saturated fatty acids of organelle and plasma membranes, are
most susceptible to free radical attack. The result of these
reactions is deleterious to the structure and function of the
affl icted cel 1 part.
Peroxidase, along with superoxide dismutase and cata-
lase, is very important as a protectant against free radicals
in the cell. These enzymes act as intracellular defense mech-
anisms which diminish effects of oxidative destruction in the
cel 1.
Superoxide dismutase is involved in the reaction to
combine superoxide free radicals to form hydrogen peroxide:
02t + 02t + 2H+ --> H202 + 02
Hydrogen peroxide (H202) is less reactive than the superoxide
free radical (02T), but it has more potential for lipid auto-
oxi dation.
Peroxidase and catalase normally degrade hydrogen pero-
xide to water in the final detoxification step:
H202 + AH2 ---> 2H20 + A
The coordinated action of peroxidase, along with superoxide
dismutase and catalase, allows aerobic cells to remove super-


oxide and hydrogen peroxide before these substances can generate
hydroxyl radicals.
^2* + OH + "OH +
The superoxide free radicals and the hydroxyl radical are both
potent oxidizing agents. If peroxide degrading enzymes are
deficient, hydrogen peroxide (^C^) and hydroxyl radicals ('OH)
would accumulate and increased oxidation could occur.
Age related changes in the activities of peroxidase,
catalase and peroxides have been demonstrated. In Drosophila
me!anogaster, a decreasing curve was found for both peroxidase
and catalase from eclosure up to 100 days. The peroxide level
was found to increase with age. Peroxidase showed a more pro-
nounced decrease with age compared to catalase. The increasing
peroxide levels corresponded more with the declining peroxidase
values than with catalase values (Armstrong et al., 1978). This
may mean that peroxidase is more important in the free radical
protectant function than catalase. It does indicate that both
catalase and peroxidase levels are changing with age.
Four major isozymes of peroxidase have been found in
both pre-eclosure and adult forms of Drosophila melanogaster.
Using spectrophotometric methods, Poole (1983) found four iso-
zyme peaks in homogenates of adult forms. Peaks of peroxidase
activity at distinct pH optima generally correspond to variant
forms of an enzyme, or isozymes. These pH optima were at
pH 5.0, 5.9, 7.4 and 8.9. Lichtenstein (1984) developed an
electrophoretic assay to look at peroxidase isozymes of single


flies. She also found four isozymes present in preadult and
adult Drosophila melanogaster. No effort has yet been made
to compare the isozymes found by spectrophotometric methods with
the electrophoretic bands.
The peroxidase isozymes observed by Lichtenstein repre-
sent a transition of the enzyme through the continuous develop-
mental process. Predominant patterns are correlated with develo
mental stages. The transition from larva to early pupa was
associated with a shift from isozymes #3 and #4 to isozymes
#1 and #2. The middle pupa had a large drop in total peroxidase
activity; thus, isozymes could not be visualized in the gels
at this stage. The late pupal stage was represented by isozymes
#3 and #4. The transition from late pupa to newly emerged
adults was.associated with a pattern shift similar to that of
larva to early pupa; a shift from isozymes #3 and #4 to iso-
zymes #1 and #2. A major question which arises from Lichten-
stein's studies is what is the source of the variability of the
isozyme patterns within a developmental stage and between
developmental stages.
Markert was one of the first to elucidate the physio-
logic significance of an enzyme in the developmental process.
The lactic dehydrogenase system is a classic example of develop-
mental control of an enzyme system. Lactic dehydrogenase (LDH)
is an enzyme that catalyzes particular steps during carbohy-
drate metabolism that affect the concentrations of the inter-
mediary compounds lactate and pyruvate.


7
There are five isozymes of LDH which differ from each
other sufficiently to be separated by electrophoresis. Markert
and Ursprung (1962) determined that the relative amounts of the
isozymes are markedly changed among different tissues, and also
that the relative proportions of the isozyme undergo significant
changes during development in any one tissue.
Developmental studies have been carried out for several
enzyme systems in Drosophila melanogaster. The activity of
alcohol dehydrogenase (ADH) has been determined at various
developmental stages. The enzyme activity starts at a low
level in embryos, begins to rise before hatching and continues
to rise during the larval stages, reaching a maximum in the
late third instar stage. The activity declines during the
pupal stage to intermediate values. In newly emerged adults,
ADH activity approximately doubles, to reach a second maximum
in adult flies (Dickinson and Sullivan, 1975).
The work on the intraspecific variation of the two major
isozyme classes, ADH-S and ADH-F, provide a good working basis
for developmental, physiologic and genetic studies of other
gene-enzyme systems. Other examples of enzymes exhibiting
developmental control include alpha-amylases, glucose-6-phos-
phate dehydrogenase, xanthine dehydrogenase, esterases and
pepti dases.
To search for genetic variation of the peroxidase enzyme,
it was decided to survey nine different strains of Drosophi1 a
me!anogaster at the developmental stage easiest to visualize


bands, the third instar larval stage. This developmental stage
lasts about two days; from approximately 69 hours to 117 hours
after egg hatching (Demerec, 1950).
Gel electrophoretic runs of homogenates of third instar
larvae were carried out. If all the strains showed the same
isozyme pattern, a purely developmental pattern would be supported.
If each strain had a distinct pattern, a genetic basis would be
indicated. These two factors, developmental stage and genetic
basis, plus environmental factors, are thought to be integrated
to determine the isozyme pattern.


CHAPTER II
MATERIALS AND METHODS
Drosophila Stocks and Homogenates
Nine separate strains of Drosophila melanogaster were
used in this experiment. These are listed in Table 1. Male and
female flies were maintained together in six ounce plastic
bottles with cardboard stoppers. Approximately 40cc of Instant
Drosophila Medium (Carolina Biological Supply 4-24), an equal
amount of deionized water and a few grains of active dry yeast
(Fleishmann) were placed in each bottle as a food source. The
stock bottles were kept at room temperature and exposed to
natural, seasonal daylight and darkness cycles, and to natural
humidity conditions.
Individual larvae were removed from the bottles by using
a wooden applicator stick. Third stage instar larvae which had
emerged from the medium and which were crawling on the sides of
the plastic bottles were used without regard to sex.
The individual larvae were placed in 20 ul of Tris-glyci
buffer, pH 8.7, + 0.5% Triton X-100 in a 0.1 ml glass tissue
grinder tube. Homogenation was performed by hand using a pestle
to fit the tube until the larva appeared to be completely homo-
genated, approximately 30 seconds. After one hour of incubation


10
the homogenate was extracted with an Eppendorf digital micro-
pipetter for measured application onto the gels.
TABLE 1
Description and Source of Drosophila melanogaster Stocks
Strain Descri ption Source
bn/st mutant strain/white eyes Carolina Biological Supply Company
B56 inbred derivative of wild National Center for
type Atmospheric Research
Oreboro wild type strain William Sofer/Waksman Institute
Bethylie wild type strain William Sofer/Waksman Insti tute
Canton classic wild type strain William Sofer/Waksman Insti tute
CL55 homozygous at all loci C. Laurie-Ahlberg/Chapel Hill, North Carolina
bcnvg black body color/cinnabar Carolina Biological
eyes/vestigial wings Supply Company
pgal n9 used in heat shock studies William Sofer/Waksman Insti tute
Adh fn23 Adh(-) mutant William Sofer/Waksman Institute
The following procedures for slide preparation, sample
preparation and gel electrophoresis used in this project were
developed by P. Lichtenstein (1984) for electrophoretic evalua-
tion of peroxidase isozymes present in single organisms. A
summary of the reagents used in the procedure are listed in
Table 2.
Procedure
Slide Preparation
Microscope slides (75x50 mm) were cleaned by soaking
in soapy water overnight. Hot water was run over the slides


n
TABLE 2
Summary of the Reagents used in this Procedure
1. Gel Buffer 43 mM Tris in 46 mM Glycine
Tris (Sigma T-1503) 5.16 gm
Glycine (Sigma G-7126) 3.5 gm
Mix to 1 liter with deionized water. pH to 8.7 with 5N
NaOH. Mix well and store at 4C.
2. Electrophoresis Buffer 10 mM Tris in 333 mM Glycine
Tris (Sigma T-1503) 1.2 gm
Glycine (Sigma G-7126) 25 gm
Mix to 1 liter with deionized water. pH to 8.7 with 5N
NaOH. Mix well and store at 4C.
3. Homogenating Buffer
Add 5 ul Triton-XlOO (Sigma T-6878) to 10 ml of Tris-Glycine
Gel Buffer. Mix well and store at 4C.
4. Mcllvaine's Buffer 88 mM Phosphate in 56 mM Citric Acid
0.2 M Disodium Phosphate (Sigma S-0876) 14.2gm/500ml 441 ml
0.1 M Citric Acid (Sigma C-7129) 12.6gm/600ml 559 ml
Mix well with specified amounts of deionized water. pH to
4.4 with concentrated HC1. Mix well and store at 4eC.
5. DAB-Gelatin Peroxidase Stain
Dissolve 100 mg Knox unflavored gelatin in 50 ml of boiling
deionized water (mix until dissolved using stir bar). When
the solution is at room temperature, add 18 mg of 3,3'-
diaminobenzidine-tetrahydrochloride Grade II (Sigma D-5637)
and mix until dissolved. Keep foil around the flask to
avoid exposure to light. Add 50 ml of Mcllvaine's Citric-
Phosphate Buffer, pH 4.4. Just prior to use, add 1.6 ml
0.6% hydrogen peroxide.
6. 0.6% Hydrogen Peroxide
Add 0.1 ml 30% Hydrogen Peroxide (Sigma H-1009) to 4.9 ml
deionized water. Mix well.
7. Agarose-Dextran Gel
1.4 gm Agarose (Biorad 162-0100)
1.0 gm Dextran T-10 (Pharmacia 17-0250-01)
Mix to 100 ml with Tris-Glycine Gel Buffer, pH 8.7.
Heat at high temperature using stir bar until dissolved
(which is shortly after it comes to a full boil).


12
for one hour, followed by cold water for one hour. The slides
were then dipped in acetone (Fisher A-18-1) and allowed to air
dry.
The cleaned slides were then dipped in a boiling solu-
tion of 0.2% agarose and allowed to dry vertically. Slides were
stored in a dust-free container. This coating allowed the
agarose-dextran gel to adhere to the glass slide which acted
as a support.
Gel Casting
1. A dust-free 0.2% agarose coated slide was labelled
using a diamond-tipped etcher on the short end of one side of
the siide.
2. With the etched side down, the slide was placed on
two applicator sticks in a large petri dish on a slide warmer.
The slide warmer was heated to approximately 60-80cC.
3. Polyethylene spacers were placed on each corner of
the siide.
4. A dust-free acetone cleaned slide was placed on the
spacers, slightly offset from the coated slide on the 50 mm edge.
5. When the slides were warm and the agarose-dextran
gel dissolved, the area between the two glass slides was filled
with the hot agarose-dextran solution using a disposable glass
pasteur pipette. The area between the slides filled by cap-
illary action. The formation of bubbles was avoided. Enough
hot agarose-dextran solution was added to fill the entire area


between the slides with a slight bulge at the edges.
6. Two sets of forceps were used to push the slides
together so all of the edges were flush. The sandwich slides
were placed in a moist box (a large petri dish with a cover with
slightly moistened #3 Whatman filter pater in the bottom), and
set in the refrigerator for one hour until the gel was set.
The slides could also be stored overnight in the refrigerator.
Sample Preparation
A single larva was placed in a tissue grinder tube.
Twenty ul of room temperature homogenating buffer was added.
The larva was homogenated using a pestle. The tubes were
covered with parafilm to prevent evaporation. The sample was
incubated for one hour at room temperature.
Sample Application
1. Sandwich slides were removed from the moist box.
Spacers were removed using forceps. The top glass slide was
gently slid off from the hardened gel (which remained attached
to the bottom slide). The gel was blotted with Whatman #1
filter paper to remove any excess moisture.
2. A template was set on the gel about one third down
from the top 50 mm edge. The template was gently tapped down
on all sides of the three application slits to ensure proper
contact wi th the gel.
3. Using an Eppendorf digital micropipetter, 5 ul of
a sample was applied to one well of the template. This was


14
incubated for thirty minutes at room temperature to ensure
sample absorption into the gel.
Preparation of the Electrophoresis Chamber
1. While samples were absorbing into the gel, the
electrophoresis apparatus was set in the refrigerator and pre-
pared. Gloves were worn during this process. Approximately
130 ml of electrophoresis buffer, pH 8.7, was poured into each
chamber. The wicks were dipped in the buffer and set on the
divider of the chamber so that one edge was in the buffer and
the other edge in the center chamber. Frozen sponges were
placed in the center chambers.
2. After thirty minutes, the templates were removed
from the gel with forceps. The slides were placed in the elec-
trophoresis chamber gel side down. The sample was placed at
the cathodic end.
3. The chamber was connected to a power supply. The
electrophoresis was run at 145 volts for 90 minutes. The current
ranged from 20 to 10 mAmps.
Preparation of the Stain
While the electrophoresis was being run, the DAB-stain
was prepared. After 90 minutes of electrophoresis, the power
was turned off and the slides removed. The slides were placed
in a plastic container and the stain was poured over the slides.
A lid was placed on the container and the entire apparatus was


15
set in a dark place. The slides were left in the stain for
about four hours, or until the bands stained. They could also
be left overnight, but the background stain was then darker.
The staining solution was poured off from the slides
and the slides then soaked in deionized water for up to 24 hours
to remove background stain. The slides were allowed to dry
verti cally.
Pattern Evaluation
The isozymes were visually inspected to determine the
number, location, identity and relative intensities of the bands.
A densitometric scan was also performed on the slides. The
large slit setting at a wavelength of 520 nm was used. The
height of each peak (isozyme) was measured in millimeters. The
relative peak height percentage was calculated for each isozyme
pattern with the following formula.
Fiactioj! Height 100 = Percent Fract1on
Sum of all
Fraction Heights


CHAPTER III
RESULTS
Approximately 12 to 16 zymograms of each strain were
performed, except for strain CL55, where 49 larvae were studied.
The isozyme bands were numbered to facilitate identification.
The least anodic migrating band was labeled #1, and the rest
were labeled #2, #3 and #4 in the order of increasing anodic
migration. A total of seven distinct isozyme patterns were
observed on the slides and by densitometric scans for third
instar larvae. These are represented in Figure 1. The quanti-
tative distribution of the patterns observed in the individual
strains are listed in Table 3.
Seventeen zymograms of pupae were performed. Early
pupae were unpigmented, newly pupated forms. Middle pupae
were pigmented pupae with no visual signs of metamorphosis.
Five of the eight early pupae homogenates were the only zymo-
grams to show visible staining. The nine middle pupae homo-
genates gave no color development on the zymograms. These
results are in Table 4.


17
Pattern
Number
'i
2
3
a
5
6
7
8
Isozyme Band
_ji_
_JL
n
aJ\
il_n_
AJ\_
f\___
FIGURE 1
Third
Instar
Larvae
Early
Pupae
Transition of Isozymes seen in Third Instar Larvae
ane Early Pupae Illustrated by Densitometric Scans


18
TABLE 3
Relative Percentage of each Isozyme within a Pattern
Determined by Densitometric Scans: Third Instar Larvae^
Pattern # N Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strain: Adh fn^3
3 2 mean = 46% mean = 54%
(s = 7.1) (s = 7.1)
4 9 mean = 28.9% mean = 41 .1% mean = 30%
(s = 15.6) (s = 6.3) (s = 9.9)
6 2 mean = 63% mean = 37%
(s = 9.9) (s = 9.9)
7 1 mean = 100%
1 No color development
Strain: B56
2 2 mean = 100%
3 1 mean = 52% mean = 48%
4 5 mean = 36.2% mean = 39.4% mean = 24.4%
(s = 14.6) (s = 7.4) (s = 8.5)
6 5 mean = 52.4% mean = 47.6%
(s = 3.6) (s = 3.6)
Strain: Bethylie
2 4 mean = 100%
3 1 mean = 46% mean = 54%
4 7 mean = 27.8% mean = 36.7% mean = 35.4%
(s = 10.9) (s 1.8) (s = 9.7)
Strain: Canton
3 1 mean = 50% mean = 50%
4 1 mean = 7% mean = 48% mean = 45%
6 13 mean = 51.5% mean = 48.5%
(s = 11.9) (s = 11.9)
2 No color development
Strain: Oreboro
1 1 mean = 100%
2 3 mean = 100%
3 3 mean = 56.3% mean = 43.7%
(s = 3.5) (s = 3.5)
4 2 mean = 23.5% mean = 52.5% mean = 24%
(s = 7.8) (s = 5.0) (s = 2.8)
6 4 mean = 41.5% mean = 58.5%
(s = 14.0) (s = 14.0)
T
s
= standard deviation


19
TABLE 3 (Continued)
Pattern # N Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strain: bcnvg
2 2 mean = 100%
3 2 mean = 39.5% mean = 60.5%
(s = 7.8) (s = 7.8)
4 6 mean = 21.8% mean = 50.3% mean = 27.8%
(s = 14.4) (s = 8.3) (s = 8.5)
6 3 mean = 43.3% mean = 56.7%
(s = 20) (s = 20)
7 2 mean = 100%
Strai n: jS gal i
4 3 mean = 32.7% mean = 39.3% mean = 28%
(s = 13.4) (s = 7.D (s = 8.2)
6 11 mean = 63.4% mean = 36.6%
(s = 13.8) (s = 13.8)
7 2 mean = 100%
3 No color development
Strain: bn/st
2 1 mean = 100%
3 2 mean = 49% mean = 51%
(s = 1.4) (s = 1.4)
4 5 mean = 23.6% mean = 43.2% mean = 33.2%
(s = 12.2) (s = 4.6) (s = 8.1)
5 3 mean = 71.3% mean = 28.7%
(s = 11.9) (s = 11.9)
6 2 mean = 67.5% mean = 32.5%
(s = 12.0) (s = 12.0)
Strain: CL55
1 5 mean = 100%
2 26 mean = 100%
3 11 mean = 50.2% mean = 49.8%
(s = 5.6) (s = 5.6)
4 7 mean = 21.4% mean = 49.3% mean = 29.3%
(s = 12.9) (s = 7.5) (s = 7.1)


20
TABLE 4
Relative Percentage of each Isozyme within a Pattern
Determined by Densitometric Scans: Early Pupael
Pattern # N Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strain: CL55
7 1 mean = 100%
8 4 mean = 55.5% mean = 45.5% (s = 3.3) (s = 3.3)
3 No color development
1
s
standard deviation


CHAPTER IV
DISCUSSION
Multi-banded enzyme systems, in which various electro-
phoretic forms appear and disappear or change in relative con-
centration as development proceeds, are good systems to use in
a study of gene regulation. One requirement of such use is
that the enzyme must be differentially expressed with respect
to either timing or localization. The changing patterns may be
attributed to changes in the relative activity of different
genes.
An isozyme is the term used for any two or more distinct
forms of an enzyme which have identical or nearly identical
chemical properties, but differ in some property such as net
electrical charge, pH optima, number and type of subunits, or
substrate concentration. Peroxidase has four known isozymes
in Drosophila melanogaster (Poole, 1983; Lichtenstein, 1984).
The results obtained from the gel electrophoretic runs
do not show one specific isozyme pattern for all of the third
instar larvae. This would tend to rule out a purely develop-
mental model (or all third instar larvae would show the same
pattern).
The predominant isozyme seen in the majority of the
strains was isozyme #3. Isozyme #2 was never visualized in


22
larval samples. Overall, isozymes #1, #3, and #4 occurred
separately or in various combinations. Examples of some of these
banding patterns are presented in Figure 2. Lichtenstein (1984)
hypothesized a trend in the larval stage of isozymes #3 and #4
present in early larval stage with #1 occurring during the
later larval stage. She also hypothesized that the transition
from larva to early pupa was associated with a shift from iso-
zymes #3 and #4 to isozymes #1 and #2.
The patterns seen in this study follow Lichtenstein's
proposed transition, except for the obvious absence of isozyme
#2. Considering that late stage larvae were used, this isozyme
should have been visualized. Electrophoretic separation tech-
niques may not have been as exact as those used by Lichtenstein.
There were several combinations of isozyme bands seen
in this study that were not reported in Lichtenstein's. These
may be due to the greater number of larvae tested and also may
be due to the separate inbred strains used. The transitions
between larval and pupal forms were not timed exactly, which
may also contribute to the additional combinations observed.
The one strain used that is homozygous at all loci is
the strain called CL55. This strain showed the most striking
results. Of the larvae tested, 86% (42/49) showed only iso-
zyme bands #3 and/or #4. Only 14% (7/49) showed a slight appear-
ance of isozyme #1 along with #3 and #4.
If the peroxidase was controlled by one codominant


23
I
A
FIGURE 2
Zymograms Example Isozyme Patterns of Third Instar
Larvae and Early Pupae
Third Instar Larvae:
A) Strain Bethylie; Pattern #2; Isozyme #4
B) Strain CL55; Pattern #3; Isozymes #3 and #4
C) Strain Adh fn23; Pattern #4; Isozymes #1, #3 and #4
D) Strain bcnvg; Pattern #4; Isozymes #1, #3 and #4
E) Strain B56; Pattern #6; Isozymes #1 and #3
Early Pupa:
F) Strain CL55; Pattern #8; Isozymes #1 and #2


24
gene locus, three patterns would be observed; AA, AB and BB.
Four patterns were seen in the CL55 strain; band 4, band 3,
bands 3 and 4, and bands 1, 3 and 4. There are a number of
possible explanations for such a pattern.
One explanation could be that this CL55 strain is not
truly homozygous at all loci. This would allow for more alleles
to be present in the population, leading to other banding
patterns. But the care taken in keeping the stocks most likely
rules out this possibility.
Another explanation could be that an environmental event
affected the phenotypic expression of the genotype. A geno-
troph displays heritable changes induced within a single com-
pletely inbreeding genotype by a single generations' growth in
contrasting environmental conditions. It has been shown that
by growing one generation of flax (Linum usitatissimum L.) in
a specific fertilizer, that the isozyme patterns from this gen-
eration and at least the following 20 generations are slightly
shifted (Tyson et al., 1985).
The isozyme #1 band was seen in runs done on June 25,
July 1 and July 2, 1986. The food medium was kept constant
throughout this experiment. The humidity and temperature con-
ditions changed daily in the laboratory, and possibly these
caused a genotrophic change. However, there was no pattern
seen of daily or seasonal change which correlated with the
presence or absence of certain bands.


25
The third instar larval stage lasts for about 48 hours.
The selection of larvae for the homogenates was not timed
exactly; they were selected by visual estimation. Larvae were
chosen if they were crawling up the sides of the bottle and
were a fairly good size. Many developmental changes occur
during the larval stage before pupae formation. Lichtenstein
(1984) saw isozyme #1 predominantly in the early pupal stage.
She defined this as an organism which had stopped moving on the
side of the bottle, but which as yet had no pupal case. So,
another possibility for the visualization of the fourth pattern
in the CL55 strain, isozymes #1, #3 and #4 is that some of these
third instar larvae could have been undergoing some develop-
mental changes to the early pupae stage already. Isozyme #1
is of interest, then, because it appears to be present in early
larvae of other strains, but not present in early larvae of
strain CL55. There could also be a developmental strain dif-
ference in CL55 which causes isozyme #1 to be slower in becoming
activated.
The strain bn/st is the only strain which had an iso-
zyme pattern of isozymes #1 and #4. These two isozymes are on
opposite ends of the transition scheme (refer to Figure 1), so
it would be expected to have an intermediary isozyme present
also. This could also be an example of a developmental strain
difference. Isozyme #1 could be activating while isozyme #4
is still degrading in strain bn/st.


\
26
If peroxidase was controlled by two codominant gene
loci, one could see six different isozyme bands in various com-
binations: AA CC, AB CC, BB CC, AA CD, AB CD, BB CD, AA DD,
AB DD, and BB DD. If the two gene loci showed complete dominance
one could see four distinct bands, but you would only see two
bands at a time: AA CC, A- CC, AA C-, and A- C-. In every
strain, a pattern was seen with three isozyme bands present at
once, namely the pattern of isozymes #1, #3 and #4. So the
regulation of peroxidase does not seem to be either of these
two models.
If each band was controlled by its own locus, there
would have to be null alleles present. If the null allele were
dominant, AA and Aa would not show up; aa would be isozyme
band #1. With this scheme there would be four bands, with
zero to all four occurring at one time. This scheme could
explain the results seen in each of the strains tested. A
similar scheme is proposed for the electrophoretic variants
in Avena fatua for peroxidase (Clegg and Allard, 1973). However,
to hypothesize null alleles for all four loci is a very compli-
cated scheme, and is not too likely.
The peroxidases represent a wide group of isozymes in
plants, which also generally show inter- and intraspecific
variability. This variability is also shown during plant de-
velopment. Almost all of the genetic studies carried out with
peroxidase in plants coincide in showing their monomeric


27
character and monogenic control, and in showing the existence
of null alleles. The peroxidase system in Drosophila melano-
gaster appears to follow some of these same trends, but it may
not be under monogenic control and it may not be a monomer.
Myeloperoxidase is a dimer and glutathione peroxidase is a
tetramer. Knowing the structure of the peroxidase enzyme in
Drosophila melanogaster will be very beneficial in studying the
genetics of the system.
Seventeen zymograms were done on homogenates of pupae
of the CL55 strain. Four zymograms of early pupae homogenates
had isozymes #1 and #2 present. Three of the eight early pupae
homogenates showed no color development. This may be due to
selection of middle pupae instead of early pupae. There was no
visible staining in the zymograms of the nine middle pupae homo-
genates. More pupae homogenates need to be tested before any
conclusions can be drawn, but the presence of isozyme #2 in the
early pupae supports the transition scheme outlined earlier.
The results serve to emphasize the importance of genetic
analysis of electrophoretic variation. Any study of enzyme
polymorphism using electrophoresis to characterize levels of
genic variation must involve genetic as well as electrophoretic
analysis before conclusions can be drawn.
Many future studies can be designed to clarify and ex-
trapolate these results. A first step would be to perform gel
electrophoresis on homogenates of third instar larvae of a


28
different completely homozygous strain. If only one isozyme band
was present, this would give support to a single gene hypothesis.
Also, if this strain had a single isozyme band present different
from strain CL55, crosses could be made. If only hybrid bands
resulted, support would be given for codominance.
A major focus in gene regulation systems is posttrans-
lational modification. It has been suggested that the absence
of codominance in F-j hybrids would suggest posttranslational
modification (Fieldes and Tyson, 1984). The peroxidase isozymes
in flax are all glycoproteins. A possible common site for a
posttranslational modification in these molecules is, therefore,
the carbohydrate moiety. The observed shifts in molecular
weight may reflect changes in molecular conformation. Differ-
ences in posttranslational modification would likely have far-
reaching effects on the organisms' physiology and phenotypic
characteristics. In the peroxidase system studied, there is not
enough information even to speculate about any posttranslational
effects.
Dietary factors have been shown to be important in
various enzyme systems. Exposure of Drosophila melanogaster
to 2-propanol results in a shift in the patterns of alcohol
dehydrogenase (ADH) isozymes toward greater amounts of ADH-1.
Administration of dietary ethanol results in a decrease in the
concentration of ADH-1 relative to the other isozymes (Laurie-
Ahlberg, 1985). This ethanol influence is affected by the


29
level of dietary carbohydrate and also differs with various
genotypes. Environmental factors and genotype-environment in-
teractions that effect enzyme activity play a major role in
understanding the regulatory system and the physiological sig-
ni ficance.
The diet was kept constant throughout this study, so it
was not a variable. Once peroxidase is better understood, diet
could be a variable in studies to test the effect on the iso-
zyme's expression.
The isozyme patterns shift throughout the development of
Drosophila melanogaster. It has been shown that the overall per-
oxidase level decreases with age, a finding which supports the
free radical theory of aging. Life span studies could be carried
out on various strains. Each strain could be timed exactly from
egg laying to adult death. Any differences in life spans between
strains could be correlated to the presence or absence of a par-
ticular isozyme band. For example, compare strain CL55, which
usually has isozymes #3 and #4 in the third instar larval stage,
to a strain like gal n^, which almost always showed isozyme #1
in the same developmental stage. If there was a major difference
in overall life spans between the two strains, it may be correla-
ted to the presence or absence of isozyme #1.
The four isozymes seen may not be the only isozymes of
peroxidase. This method detects only the active isozymes. In
the adult fly, peroxidase is attached to protein carriers.


30
It is possible that there are other isozymes undetected in the
adult stage because it is difficult to get the enzyme free.
These may be dormant in the pre-adult stages, so they are not
detected by electrophoretic methods either.
All of the isozymes, the four known ones and any unknown,
are important and have a physiologic significance. Some are
important in the aging process, others may not be. Of the four
isozymes Poole (1983) found spectrophotometrically, only two
showed a decrease with age.
The results compiled from this study are based on much
individual interpretation. The isozyme patterns observed were
basically the same as Lichtenstein's (1984), but there was a
lot of variability in the intensity of staining. The large
standard deviations are due to the small sample sizes.
This study reproduced and confirmed many of the findings
of previous work (Lichtenstein, 1984), particularly the transi-
tions of isozymes during the larval stage. In addition, inter-
strain developmental differences have been pointed out by the
use of inbred strains.
Further studies will build and extrapolate on this in-
vestigation to elucidate the basis for regulation and the develop-
mental importance of the peroxidase system in Drosophila melano-
gaster.


BIBLIOGRAPHY
Armstrong D., R. Rinehart, L. Dixon, and D. Reigh.(1978).
Changes of peroxidase with age in Drosophila. Age.
1:8-12.
Board P. G. (1983). Further electrophoretic studies of ery-
throcyte glutathione peroxidase. Am. J. Hum. Genet.
35:914-918.
Cavallo T. (1976). Cytochemical localization of endogenous
peroxidase activity in renal medullary collecting
tubules and papillary mucosa of the rat. Laboratory
Investigation. 34:223-227.
Clegg M. T. and R. W. Allard (1973). The genetics of electro-
phoretic variants in Avena. II. The esterase E], E2,
E4, E5, Eg and anodal peroxidase APX4 loci in A. fatua.
Journal Heredity. 64:2-6.
Demerec M. (1950). Biology of Drosophila. Hafner Publishing
Company, New York.
Dickinson W. J. and D. T. Sullivan (1975). Results and problems
in cell differentiation a series of topical volumes
in developmental biology, p. 70-78, in Beerman W., J.
Reinert and H. Ursprung (ed.) Gene-Enzymes Systems in
Drosophila. Springer-Verlag, New York.
Edwards H. H. and M. Morrison (1976). Localization of thyroid
peroxidase and the site of iodination in rat thyroid
gland. Biochem. J. 158:477-479.
Espino F. J. and A. M. Vasquez (1982). Genetic control and
modifications of peroxidase pattern in two species of
Nicotiana and their interspecific hybrid. Experientia.
38:321-322.
Fieldes M. A. and H. Tyson (1984). Possible posttranslational
modification, and its genetic control, in flax genotroph
isozymes. Biochemical Genetics. 22:99-114.
Harman D. (1956). Aging: A theory based on free radical and
raidation chemistry. J. Gerontol. 11:298-300.


32
Laurie-Ahlberg C. C. (1 985.). Genetic variation affecting the
expression of enzyme-coding genes in Drosophila: An
evolutionary perspective. Isozymes: Current Topics
in Biological and Medical Research. 12:33-88.
Lehninger A. L. (1975). Biochemistry. Worth Publishers, Inc.,
New York.
LiBassi R. H. (1981). Localization and feeding effects of
peroxidase in Drosophila melanogaster. Thesis submitted
to the Faculty of the Graduate School of the Univer-
sity of Colorado, Department of Biology.
Lichtenstein P. S. (1984). Detection of peroxidase isozymes
from Drosophila melanogaster by zymogram and crossed
immunoelectrophoresis^Developmental changes. Thesis
submitted to the Faculty of the Graduate School of the
University of Colorado, Department of Biology.
Locke M. (1969). The localization of a peroxidase associated
with hard cuticle formation in an insect, Calpodes
ethlius stroll, Lepidoptera hesperiidae. Tissue and
CeTT. 1 :555-574.
Markert C. L. and H. Ursprung (1962). The ontogeny of isozyme
patterns of lactate dehydrogenase in the mouse. Dev.
Biol. 5:363-381.
Matheson N. R., P. S. Wong, and J. Travis (1981). Isolation
and properties of human neutrophil myeloperoxidase.
Biochemistry. 20:325-330.
Poole J. H. (1983). Peroxidase isozymes in Drosophila melano-
gaster : Enzymatic properties, activity and proposed
functions during development and aging. Thesis sub-
mitted to the Faculty of the Graduate School of the
University of Colorado, Department of Biology.
Stafford H. A. and S. Bravinder-Bree (1972). Peroxidase isozymes
of first internodes of sorghum. Plant Physiol.
49:950-956.
Sunde R. A. and W. G. Hoekstra (1980). Structure, synthesis
and function of glutathione peroxidase. Nutr. Rev.
38:265-273.
Tyson H., M. A. Fieldes, C. Cheung, and J. Starobin (1985).
Isozyme relative mobility (Rm) changes related to leaf
position; Apparently smooth Rm trends and some impli-
cations. Biochemical Genetics. 23:641-654.


APPENDIX
Raw Data for Table 3: Percentage of each Isozyme Determined
by Densitometric Scans
Pattern # Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strain: Adh fn23
3 41% 59%
3 51% 49%
4 29% 38% 33%
4 6% 48% 46%
4 46% 33% 21%
4 39% 37% 24%
4 18% 47% 35%
4 54% 33% 13%
4 16% 45% 39%
4 19% 49% 32%
4 33% 40% 27%
6 56% 44%
6 70% 30%
7 100%
No color development 1
Strain: B56
2 100%
2 100%
3 52% 48%
4 29% 43% 28%
4 21% 45% 34%
4 47% 30% 23%
4 28% 46% 26%
4 56% 33% 11%
6 53% 47%
6 54% 46%
6 46% 54%
6 55% 45%
6 54% 46%


34
Raw Data for Table 3: Continued
Pattern # Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strain: Bethylie
2 100%
2 100%
2 100%
2 100%
3 46% 54%
4 43% 33% 24%
4 16% 37% 47%
4 13% 38% 49%
4 32% 38% 30%
4 36% 36% 28%
4 23% 38% 39%
4 32% 37% 31%
Strain: Canton
3 50% 50%
4 7% 48% 45%
6 29% 71%
6 41% 59%
6 59%. 41%
6 69% 31%
6 64% 36%
6 44% 56%
6 52% 48%
6 67% 33%
6 48% 52%
6 52% 48%
6 53% 47%
6 56% 44%
6 36% 64%
No color development - 2
Strain: Oreboro
1 100%
2 100%
2 100%
2 100%
3 53% 47%
3 60% 40%
3 56% 44%
4 18% 56% 26%
4 29% 49% 22%
6 46% 54%
6 25% 75%
6 37% 63%
6 58% 42%


35
Raw Data for Table 3: Continued
Pattern i # Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strai n: bcnvg
2 100%
2 100%
3 45% 55%
3 34% 66%
4 30% 50% 20%
4 23% 54% 23%
4 6% 52% 42%
4 18% 51% 31%
4 9% 60% 31%
4 45% 35% 20%
6 44% 56%
6 53% 37%
6 23% 77%
7 100%
7 100%
Strai n: ft gai n9
4 23% 47% 30%
4 48% 33% 19%
4 27% 38% 35%
6 62% 38%
6 73% 27%
6 76% 24%
6 31% 69%
6 65% 35%
6 55% 45%
6 70% 30%
6 75% 25%
6 69% 31%
6 73% 27%
6 48% 52%
7 100%
7 100%
No color development 3


36
Raw Data for Table 3: Continued
Pattern # Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strain: bn/st
2
3
3
4 26%
4 19%
4 10%
4 43%
4 20%
5 66%
5 63%
5 85%
6 76%
6 59%
Strain: CL55
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
100%
50% 50%
48% 52%
40% 34%
47% 34%
48% 42%
37% 20%
44% 36%
34%
37%
15%
24%
41%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%


37
Raw Data for Table 3: Continued
Pattern # Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strain: CL55 Continued
2 100%
2 100%
3 54% 46%
3 43% 57%
3 52% 48%
3 39% 61%
3 47% 53%
3 51% 49%
3 56% 44%
3 47% 53%
3 57% 43%
3 53% 47%
3 53% 47%
4 8% 53% 39%
4 39% 41% 20%
4 10% 62% 28%
4 37% 40% 23%
4 17% 51% 32%
4 12% 51% 37%
4 27% 47% 26%
Raw Data for Table 4: Percentage of each Isozyme Determined
by Densitometric Scans

Pattern # Isozyme #1 Isozyme #2 Isozyme #3 Isozyme #4
Strain: CL55
7 100%
8 60% 40%
8 52% 48%
8 55% 45%
8 55% 45%
No color development - 3